PROJECT SUMMARY Ischemic heart disease, including myocardial infarction (MI), is the leading cause of morbidity and mortality worldwide. Neovascularization, the formation of new blood vessels, is fundamental to cardiac repair and regeneration after MI. Neovascularization proceeds by angiogenesis , i.e., vascular endothelial cells (EC) sprouting and outgrowth, and by vasculogenesis , i.e., de novo vessel generation by recruited vascular stem cells (VSC) from circulation. Both of these migratory processes are primarily stimulated by ischemia-inducible vascular endothelial growth factor (VEGF). Our preliminary data reveal a new regulatory mechanism for vascularized EC and VSC migration in response to VEGF, mediated by phosphorylation of actin-binding profilin-1 (Pfn-1). Vascular lineage-specific knock-in of phosphorylation- dead Pfn-1Y129F mutant in mice demonstrates that Pfn-1 phosphorylation is critical for ischemia-induced neovascularization in the hindlimb. Deficiency in Pfn-1 phosphorylation inhibits EC sprouting in the aorta and wound-induced neovascularization, and also suppresses VSC homing to the ischemic hindlimb, suggesting a critical role of Pfn-1 phosphorylation in both angiogenesis and vasculogenesis. Mechanistic studies show that Pfn-1 phosphorylation increases Pfn-1 binding to G-actin, and promotes actin polymerization and cell migration. Interestingly, Pfn-1 phosphorylation is directly induced by VEGFR2/Src, independent of classic PI3K-mediated multistep signal cascades. Furthermore, Pfn-1 phosphorylation is robustly and preferentially induced in the capillaries of infarcted cardiac tissue in human MI patients. Based on these findings, I hypothesize that Pfn-1 phosphorylation represents a novel, critical regulatory node in neovascularization during tissue repair and regeneration after myocardial infarction (MI). To test this hypothesis, I propose to investigate two specific aims: 1) To determine the role of Pfn-1 phosphorylation in neovascularization during tissue repair and regeneration after MI. We will determine the role of Pfn-1 phosphorylation in EC-mediated angiogenesis as well as in VSC homing to the ischemic heart and sequent vasculogenesis in a murine myocardial infarction model. We will also test experimental therapy with a phosphorylation-stimulatory peptide for treating MI. 2) To determine the regulatory mechanism of VEGF- induced Pfn-1 phosphorylation. I will determine the domain(s) and specific site(s) of VEGFR2/Src for Pfn-1 phosphorylation. I will determine the regulatory mechanism by identifying potential protein-binding partner(s) and tyrosine phosphatase(s). These studies will provide important insight into the mechanisms controlling post-MI neovascularization, and may lead to the development of novel therapeutic approaches for treating ischemic heart disease in humans.